Crystallographic studies on apocarboxypeptidase A and the complex with glycyl-L-tyrosine.

Abstract

The crystal structures of zinc-free carboxypeptidase A (apocarboxypeptidase A) and the complex of glycyl-L-tyrosine with apocarboxypeptidase A are described and compared to the corresponding structures of the zinc-containing enzyme. Only small conformational changes in the zinc ligands accompany removal of the metal. Interactions between the tyrosine residue of glycyl-L-tyrosine and apoarboxypeptidase A are similar to those observed in the complex with the holoenzyme. However, in the absence of zinc, the carbonyl oxygen of the glycyl moiety now receives a hydrogen bond from the side chain of arginine-127. Although not as yet observed, a similar shift of the carbonyl oxygen of a susceptible bond from the zinc to arginine-127 could stabilize tetrahedral intermediates generated during the hydrolysis of substrates by carboxypeptidase. (not the zinc) functions as the binding site for the carboxylate group of the peptide during the hydrolytic step. Instead, the zinc serves to polarize the carbonyl group of the susceptible bond and possibly as the source of a zinc-hydroxyl (or zinc water) species for nucleophilic attack on the substrate during a deacylation step (8,9).Removal of the zinc yields an inactive enzyme, apocarboxypeptidase A (apo-CPase A) (10). The conformation of apo-CPase A appears to be similar to that of the native enzyme (11). Zinc is bound quite rapidly by the apoenzyme, with concomitant recovery of activity. Moreover, the recombination of zinc with apo-CPase A is inhibited by peptides, suggesting that these ligands may bind to the apoenzyme (12). The location of bound peptides at the active site in apo-CPase A was demonstrated by difference Fourier studies at low resolution of the complex of apo-CPase A with the dipeptide glycyl-L-tyrosine (Gly-Tyr) (13).X-ray crystallographic methods are well suited for investigating the structural consequences of zinc removal on the active site conformation and ligand-binding properties of CPase A. Crystal structures of CPase A in the native state and complexed to a variety of ligands have been described (14-16). Direct removal of zinc from crystals of CPase A yields crystals of apo-CPase A that are isomorphous with those of the native enzyme (11). Consequently, sensitive difference Fourier techniques may be used to study the structural changes accompanying zinc removal from CPase A. In this paper, we compare the structures of apo-CPase A and the complex of Gly-Tyr with apo-CPase A to the corresponding forms of the zinc forms. The implications of these structures for the binding and cleavage of substrates by CPase A are discussed. MATERIALS AND METHODSBovine CPase A, (Cox), 8-hydroxyquinoline-5-sulfonate (HQSA), and Gly-Tyr were purchased from Sigma and used without further purification. Crystals of CPase A were grown by dialysis of CPase A solutions against 0.2 M LiCl/0.02 M Tris-HCl, pH 7.5, at 40C (11). apo-CPase A was prepared by the standard procedure of soaking the crystalline enzyme in a solution containing chelator (17, 18). Although o-phenanthrol...

A Fast Flexible Docking Method using an Incremental Construction Algorithm

“…All but one of the experimentally observed hydrogen bonds have been reproduced in this solution. The one bond not realized is the interaction between the (Rees & Lipscomb, 1983) 0.17 1.7 × 10 5 XVII 2ctc (Teplyakov et al, 1993) 0.23 4.5 × 10 2 XVIII 4phv (Bone et al, 1991) 0.69 1.1 × 10 13 XIX 121p (Krengel, 1991) 0.54 2.6 × 10 6…”

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

“…Substrate binding through an induced-fit mechanism involving the equivalent of Tyr341 in pancreatic carboxypeptidases has previously been suggested. 34 Apparently, in TAFI the region involved in the induced-fit mechanism has been extended to encompass a much larger region of the protein.…”

“…It also contains Asp348, which determines substrate specificity of TAFI and Tyr341 that is strictly conserved in all carboxypeptidases and is thought to stabilize the carboxylate of the terminal residue of the substrate. 34 The dynamic flap interacts with the activation peptide through hydrophobic interactions between Tyr341 and residues Val35 plus Leu39 ( Figure 3B). The observed differences in dynamics of the flap region in the 3 molecules in the asymmetric unit most likely result from crystal packing effects.…”

“…Substrate binding through an induced-fit mechanism involving the equivalent of Tyr341 in pancreatic carboxypeptidases has previously been suggested. 34 Apparently, in TAFI the region involved in the induced-fit mechanism has been extended to encompass a much larger region of the protein.TAFIa inactivation is accompanied by a biphasic decrease in Trp fluorescence. 6,36 Consistent with this observation, unfolding and/or dislocation of the dynamic flap would significantly change the environment and, consequently, fluorescence of Trp350 and most likely Trp153, which is completely buried within the core of the catalytic domain.…”

Binding of a possible transition state analogue to the active site of carboxypeptidase A.

“…The hydrolytic mechanism of CPA has received considerable kinetic and structural study, and the enzyme has been the subject of many recent reviews (4)(5)(6)(7)(8). Important residues for substrate binding and catalysis are arginine-145, tyrosine-248, glutamate-270, and Zn(II); a catalytic role for arginine-127 is also considered in light of its interaction with a substrate in the x-ray structure of an apoenzyme complex (9). However, important details of the hydrolysis remain as yet unclear.…”